By Richard Jefferys, Project Coordinator at Treatment Action Group (TAG)

This past Wednesday, September 28, the Spanish Superior Scientific Research Council (CSIC, Consejo Superior de Investigaciones Científicas) issued a press release about the results of a phase I HIV vaccine trial involving 30 uninfected volunteers. The release refers to two recently published papers, one made available online by the journal Vaccine on September 9, the other by J. Virology on August 24 (abstracts and links below). The trial used a very common vector, modified vaccinia Ankara strain (MVA, a highly attenuated member of the poxvirus family), to deliver HIV antigens, and studied the resulting immune responses (24 participants received vaccine, 6 placebo). The results were a little better than those obtained with some other MVA vectors, but similar to responses seen with adenovirus-based vectors like the Ad5 HIV vaccine developed by Merck, which failed to show any efficacy at either preventing or controlling HIV infection in a widely-publicized phase IIb trial (the STEP study).

So far, so average. But the press release went a little overboard in attempting to sell the results to the media, triggering some of the most woefully misleading and erroneous coverage of HIV vaccine research in recent memory. Here are just a few examples of the headlines:

In isolation, the missteps in the press release are not particularly or uniquely heinous: the headline is "MVA-B Spanish HIV vaccine shows 90 percent immune response in humans." This refers to cherry-picked data derived from an analysis of either CD4 or CD8 responses to HIV antigens in a subset of 13 vaccine recipients described in the J. Virology paper; these individuals were selected because they attended a trial site in Madrid that could quickly deliver the fresh blood samples needed for the particular assay used (intracellular cytokine staining). Similarly, the press release trumpets an 85% response rate at week 48 of the study that is also derived from this 13-person subset. The Vaccine paper reports an analysis of CD4 or CD8 T cell responses in the full cohort of vaccine recipients (minus two individuals lost to follow up) at week 48 using an ELISPOT assay; the result is less impressive-sounding at 68% (15/22). The press release should have been upfront about this of course, but it’s not unusual for researchers to try and put their best-sounding data forward.

Two other missteps in the release make key contributions to the maelstrom of misinformation. The first is that the senior author of the papers, Mariano Esteban, is quoted as saying:

"MVA-B vaccine has proven to be as powerful as any other vaccine currently being studied, or even more"

In the original Spanish release, "powerful" is "potente," and I think it’s reasonable to assume that what Esteban was trying to convey is that the vaccine is as immunogenic as any other candidate out there, which isn’t an unreasonable claim. But since immunogenic is an obscure technical term for the ability of a vaccine to induce immune responses (without regard to whether there is any evidence that those immune responses are effective), a more understandable word was substituted—unfortunately one that could easily be misinterpreted as implying powerful efficacy.

Lastly, the release concludes:

"According to CSIC's researcher: 'If this genetic cocktail passes Phase II and Phase III future clinic trials, and makes it into production, in the future HIV could be compared to herpes virus nowadays'. Virus would not cause a disease anymore and would become a minor chronic infection, which would only show its effects in a low defence scenario, with a much lower contagious profile."

This is perhaps an attempt to paint an optimistic picture of what a vaccine that controlled but did not prevent HIV infection might achieve, and it does include an "if," but it is nevertheless so ripe for misinterpretation as to be foolhardy at best, and irresponsible at worst. Combined with the other potentially misleading elements of the press release, it created a perfect storm of hyperbole. Even an attempt to explain how memory T cells work became grist for misinformed articles, because many journalists are unfamiliar with the subject and thus thought that what was being described was something unique to the MVA-B vaccine (rather than just a description of memory immune responses similar to those that failed to work effectively against HIV in the STEP trial).

What makes this incident particularly dispiriting is that there is abundant background information on MVA vectors and HIV-specific immune responses available on the internet and easily accessible to any journalist who might—perish the thought—have been inclined to evaluate the CSIC release with a critical eye. Even on the simplest level, anyone writing about science should know that a 30-person phase I HIV vaccine trial can say nothing about potential efficacy.

When it comes to what the MVA-B papers actually show, they provide some evidence that the way that the CSIC researchers inserted HIV antigens into the vector may have served to increase the CD8 T cell response rate compared to other MVAs, although the CD4 T cell responses are poorer. There is no assessment of whether the CD8 T cell responses induced by MVA-B can actually kill HIV-infected cells, a parameter that has emerged as critically important for T cell-based vaccines (as Professor Esteban points out in his comment below, CD8 T cells from HIV-positive individuals primed in a lab dish by dendritic cells infected by MVA-B have been shown to kill HIV-infected CD4 cells, but it is not yet known if the same holds true for CD8 T cell responses induced by the vaccine in people). In the paper in Vaccine, the authors themselves note that immune responses are very similar to those induced by Merck’s failed Ad5-based candidate, both in terms of response rate and magnitude. Binding antibody responses are not notably different from other vaccines, and a single assay that is used to look for neutralizing antibodies only measures activity against a relatively easy-to-neutralize ("tier 1") HIV isolate named BX08; the same isolate is also the source of the HIV Env antigen used in the vaccine. Even under these artificially ideal circumstances, neutralizing antibody activity is only detected in a third of vaccine recipients.

Ultimately, in stark contrast to the wildly exaggerated claims now circulating globally, the results indicate that MVA-B will join a large group of similar poxvirus vectors that are queuing up to be considered for preventive efficacy studies. The most optimistic view is that these candidates might be able to improve on the 31% protective efficacy reported in the RV144 vaccine trial in Thailand, but there is no indication or expectation that they will render HIV as easily controlled as herpes. Therapeutic results obtained with similar vaccines have, to date, been underwhelming. There are two ongoing trials evaluating the therapeutic potential of MVA vectors in HIV-positive people, one in the UK involving a candidate developed by Oxford University, the other in the US using a DNA/MVA prime-boost approach designed by GeoVax. Further information on the HIV vaccine pipeline can be found in TAG’s annual pipeline reports and on the AVAC website.

UPDATE 10/7/2011: Professor Mariano Esteban has kindly submitted a response regarding these criticisms of the media coverage relating to MVA-B, see the comments section below. The CSIC has also updated their press release to add some caveats, along with additional information and resources for the media; the potentially misleading reference to herpes infection has been removed. It is worth noting that the unfortunate outbreak of hype around the phase I results should not be interpreted to mean that the research has no value, as there are compelling reasons to try and iteratively improve the immunogenicity of MVA vectors and Professor Esteban's research group and CSIC have made potentially important progress on that front.

RESULTS: A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers.

CONCLUSIONS: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497.

Attenuated poxvirus vectors expressing HIV-1 antigens are considered promising HIV/AIDS vaccine candidates. Here we described the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time post-vaccination as detected by IFN- ICS assay. The CD4+ T cell responses were predominantly Env directed, whereas the CD8+ T cell responses were similarly distributed against Env, Gag and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination and the responses were sustained (84.6% at week 48). Vaccine-induced CD8+ T cells to HIV-1 antigens after one year were polyfunctional and mainly distributed within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Anti-vector T cell responses were mostly induced by CD8+ T cells, highly polyfunctional and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered as promising HIV/AIDS vaccine candidate.

A new paper in the journal AIDS Research and Human Retroviruses reports that the majority of CD4 T cells harboring latent HIV infection are specific for HIV antigens (as opposed to other common antigens such as PPD, CMV or influenza). Conducted by Ashwini Shete and colleagues from the National AIDS Research Institute in India, the study investigated the capacity of a variety of antigens to stimulate HIV replication in CD4 T cells from people with HIV (both on and off ART). In untreated individuals, Env and Pol antigens invoked significantly more HIV expression than other HIV and non-HIV antigens. In a cohort of 18 individuals on ART, HIV replication was induced significantly more frequently after stimulation with Env, Pol and Gag compared to other HIV and non-HIV antigens. Notably, a minority of participants did show evidence of lower-frequency infection of cells specific for peptides from PPD, influenza, EBV and CMV. The findings echo and extend those reported in 2002 by Audreya Demoustier and colleagues (available in full text online), which documented preferential stimulation of latent HIV with a less diverse array of viral antigens (p24 and Nef).

As the authors note, these data imply that therapeutic HIV vaccines—when combined with ART—may have the potential to both activate latent viral reservoirs and bolster the immune response against the virus. The data further suggest that therapeutic HIV vaccine candidates will need to include a broad array of HIV antigens if the goal is to maximize the activation of latent HIV.

Coincidentally, the first published study to address the impact of therapeutic vaccination on the latent HIV reservoir just appeared online in the journal AIDS. Deborah Persaud and colleagues analyzed data from a pediatric trial of MVA and Fowlpox-based therapeutic HIV vaccines, finding evidence of a significant but transient reduction in the number of latently infected CD4 T cells. The vaccines contained HIV env, gag, tat, rev, nef and reverse transcriptase genes; immunizations were with MVA at weeks 0 and 4 and Fowlpox at weeks 8 and 24 (although only 11 of the 18 study participants received the final Fowlpox boost due to an interruption in supply of the vaccine). A significant reduction in the frequency of latently infected CD4 T cells was demonstrated at week 40, but was no longer detectable by week 72 of follow up. The authors state: “This effect may result from enhanced targeting of latently infected CD4 T cells by vaccine-induced HIV-specific immune responses upon exiting the latent reservoir during activation; or by direct reactivation of HIV-specific memory CD4 T cells harboring latent HIV with therapeutic immunizations, and warrants evaluation in other studies.” An ongoing trial named EraMune02 is currently exploring the impact of therapeutic vaccination on HIV reservoirs in greater detail.

Therapeutic vaccinations using Human Immunodeficiency Virus (HIV) antigens in HIV infected patients on antiretroviral therapy (ART) have so far been attempted with the purpose of inducing CTL response. However they can also be useful as a strategy for activation of latent HIV reservoir which is thought to be mainly comprised of latently infected HIV specific memory CD4 cells, eventually leading to elimination of the virus. The present study was carried out to explore ability of different HIV antigens to activate HIV replication as assessed by intracellular P24 detection as well as to induce T cell responses in terms of cytokine expression by flow cytometry after stimulation of PBMCs from HIV infected patients. HIV antigens were found to be able to activate most of the CD4 T cells harboring proviral DNA. HIV-1 Pol and Env were responsible for induction of higher HIV replication in terms of both magnitude and frequency followed by Gag and Nef. As against this, Pol and Env contributed to less number of polyfunctional CD8 cells desirable for elimination of HIV infected cells in comparison to Gag and Nef. Thus HIV antigens may provide strategy for activation of latent reservoir. It was observed that HIV replication started as early as half an hour after in vitro activation indicating stringent need for maintaining effective concentrations of antiretroviral drugs to prevent further spread of HIV during this process. HIV infected cells were found to be responsible for higher IL-10 secretion after activation, which could also serve as one of the reasons for suppressed CD8 responses to Pol and Env as more number of HIV infected CD4 cells would be secreting IL-10 in response to these antigens. Since IL-10 blockade helped in improving immune responses in terms of cytokine secretion, it should be considered in settings of therapeutic vaccination to improve CTL responses, which will ultimately limit persistence of viral reservoir.

aJohns Hopkins University School of Medicine, Baltimore, MD, United States bPediatrics and Molecular Medicine, University of MA Medical School, Worcester, MA, United States cStatistical and Data Analysis Center, Harvard School of Public Health, Boston, MA, United States dColumbia University Medical Center, NY, United States eDuke University Medical Center, Durham, NC, United States.

The effect of therapeutic immunization with modified vaccinia Ankara and Fowlpox-based HIV vaccines on the latent reservoir was examined in 19 young adults who were receiving effective antiretroviral therapy. Correlations between size of the reservoir (measured in infectious units per million [IUPM]) resting CD4+ T cells and HIV-1 specific immune responses, including immune activation were examined. Decay of the reservoir was assessed using random-effects model.

RESULTS:

A modest transient decrease in the size of the reservoir was observed at week 40 (mean -0.31-log10 IUPM [95% CI: -0.60 to -0.03; P = 0.03) following HIV vaccinations. The estimated half-life (T1/2) of the reservoir during the 40 weeks following vaccination was 9.8 months and statistically different from zero (P = 0.02), but 35.3 months and not different from zero (P = 0.21) over 72 weeks of study. Latent reservoir size at baseline was not correlated with HIV-specific CD4+, CD8+ responses or immune activation, but became correlated with CD4+ IFNγ (r = 0.54, P = 0.02) and IL-2- responses at six weeks post immunization (r = 0.48, P = 0.04).

CONCLUSION:

Therapeutic HIV-1 vaccinations led to a transient increase in decay of latently infected CD4+ T cells. Further studies of therapeutic HIV vaccines may provide important insights into facilitating decay of the latent reservoir.

Appended below is a highly subjective selection of highlights, including new data on the role of CD4 T cell responses in control of HIV, an intriguing study linking the nucleotide composition of lentiviruses to their pathogenicity, and results from animal modelers attempting to recapitulate both the recent RV144 vaccine trial in Thailand and the STEP trial of Merck’s now-discontinued HIV vaccine candidate. Links to e-Posters are included if they are available on the conference website.

Background: A combination vaccine based on the recombinant canarypox vector ALVAC-HIV and HIV gp120 envelope glycoprotein protected nearly one third of vaccinees from HIV acquisition but afforded no protection from CD4+ T-cell loss or high virus levels in vaccines that became infected in the RV144 trial in Thailand. This result was surprising, given the limited ability of either vaccine component to induce CD8+ T-cell responses or elicit broadly neutralizing antibodies. An animal model able to predict HIV vaccine efficacy for humans could hasten progress in our understanding of the immune responses that contribute to protection.

Methods: We vaccinated Indian rhesus macaques with an ALVAC-SIV and SIVgp120 immunization regimen that mimics the RV144 trial. At the end of the immunization regimen, the vaccinated animals received a titered mucosal challenge with a dose of SIVmac251 shown to transmit a limited number of variants to naive controls, recapitulating human mucosal HIV transmission.

Results: Vaccine immunogenicity and efficacy in macaques were similar to observations in humans; as in RV144, one third of vaccinated macaques were protected from acquisition SIVmac251. Viral load and CD4+ T cell numbers in animals that did become infected were not different from controls. Animals protected from infection had equivalent T-cell responses but higher avidity binding antibodies to gp120 compared to unprotected animals.

Conclusion: Titered intrarectal challenge of macaques with SIVmac251 shows potential to accurately model clinically observed vaccine efficacy, suggesting a role for this model in understanding protective responses elicited by HIV preventive vaccines. Consistent with the results from RV144, our findings suggest non- neutralizing antibody responses may play a role in protection from SIV/HIV acquisition.

Background: During acute HIV-1 infection, early control of vi- remia and establishment of viral set point have been widely attributed to HIV-specific CD8 T-cell responses. However, despite increasing evidence for direct antiviral activity by CD4 T-cells in other infections, the impact of HIV-specific CD4 T-cells on viral control has not been studied.

Methods: We studied 26 acutely infected, treatment-naive subjects and monitored their clinical outcome for up to 3 years post- infection. Baseline HIV-specific CD4 T-cell responses were assessed for degranulation (CD107a), IFNc secretion, expression of granzymes (GrzA,B,K) and perforin. HIV-specific cytolytic CD4 T-cell responses were investigated longitudinally in a subset of 11 subjects with similar peak viral loads for 1 year post-infection.

Results: Among the patients followed longitudinally, 6 progressed to a low viral set point, while 5 progressed to a significantly higher set point (134,020 vs. 11,234 copies/ml; p = 0.004). Interestingly, a significant expansion of HIV-specific cytolytic CD4 cell responses was observed in individuals who controlled viral replication compared to those who progressed to a high viral set point (IFNg: p = 0.038, CD107a: p = 0.042). Importantly, this expansion was observed early post-infection, prior to the divergence of viral load or CD4 T cell counts between the two groups. Examination of the baseline HIV-specific CD4 responses revealed a distinct GrzA-enriched cytolytic phenotype that was highly associated with subsequent viral control. Strikingly, Kaplan-Meier analysis of only the baseline cytolytic CD4 T-cell response in a larger cohort demonstrated that individuals with HIV-specific GrzA+ responses remained off HAART significantly longer than individuals with HIV-specific GrzA-responses (p = 0.0023).

Conclusion: Here we demonstrate that the rapid induction and expansion of a distinct cytolytic CD4 T-cell response during acute infection is significantly associated with viral control and disease outcome. These data suggest a pivotal role for HIV-specific cytolytic CD4 T-cells in the early control of viremia following acute infection and for future HIV vaccine design strategies.

P17.32 LB

Primary Immune Responses to Vaccinia Virus Vaccination: The Role of Cytotoxic Effector CD4+ T Cells in the Generation of Human T Cell Memory

Background: To understand the developmental stages of CD4+ T cell responses to viral infection and their differentiation into long-term memory cells, we studied individuals receiving primary vaccinia virus (VV) vaccination. This provides an attractive model for the study of human antiviral T cell responses as vaccination results in an acute infection that is cleared and leads to long-term protective immunity.

Conclusion: Generation of anti-viral human CD4+ T cell memory during primary immune responses is poorly understood. The role of CTL associated genes in this process has not been explored. Understanding their role in the generation of an effective memory may lead us closer to the development of a more effective HIV vaccine, such as enhancing the modestly efficacious HIV-1 vaccine used in the RV144 study.

Background: One of the foremost challenges in the development of an HIV-1 vaccine is that HIV-1 targets CD4-T cells, which are critically important in shaping the immune response to infection. It has been demonstrated that T follicular helper (TFH) CD4-T cells are pivotal for the induction of broadly neutralizing antibody (nAb) responses and for the generation of long-lived B-cell memory maturation. By cognate interaction and cytokine secretion, CD4-T cells expressing CD40L can induce class switching and somatic hypermutation in antigen specific B-cells, which is reflected in the expression of activation-induced cytidine deaminase (AID). Nonetheless, the role of TFH cells and their interaction with B-cells in HIV infection is currently unknown.

Methods: Ten subjects were identified during acute HIV infection and followed longitudinally over a median of three years. Neutralizing activity of plasma antibodies against a panel of present and past autologous and heterologous viruses was assessed using a single-replication cycle assay in which full-length envelope genes were incorporated into expression vectors (Monogram Biosciences). Phenotypic and functional characteristics of HIV-specific CD4-T cell responses and their impact on AID expression were assessed longitudinally by multiparameter flow cytometry.

Results: The breadth of the broadly neutralizing antibody response to heterologous HIV significantly increased over time in each patient. Interestingly, the expansion in heterologous breadth was not only significantly correlated with an increase in AID expression in B-cells, but also with CD4-T cell expression of PD-1, a marker that has been associated with TFH cells. Moreover, a positive association between AID expression and gp120-specific CD40L-responses was detected at the earliest time point.

Conclusion: Our data strongly indicate that the induction of broadly neutralizing antibody responses is linked to the function and activation of HIV-specific CD4-T cell responses. These findings will be pivotal in efforts to generate neutralizing antibody responses by vaccination strategies.

1Emory University, Atlanta, USA; 2Laboratory of Molecular Microbiology, National Institutes of Health, Bethesda, USA; 3Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, USA; 4School of Medicine, UT Health Science Center at San Antonio, San Antonio, USA; 5Department of Biostatistics, University of Pennsylvania, Philadelphia, USA; 6The AIDS and Cancer Virus Program, The National Cancer Institute, Frederick, USA

Background: The relative contribution of CD4+ T-cells to the control of virus replication during primary SIV infection rhesus macaques (RM) remains incompletely defined. To investigate the relationship between CD4+ T-cells and post-peak decline of viremia, we depleted CD4+ lymphocytes from non- inductive immunological sites of five RMs prior to SIV-infection.

Results: At the time of infection, CD4+ lymphocyte-depleted RM showed ten-fold lower CD4+ T cell levels in blood and LNs and normal CD4 + T-cell levels in RBs. After infection, CD4+ lymphocyte-depleted animals exhibited a similar peak of viremia but, in contrast to non-depleted RMs, showed no post-peak virus decline, and progressed more rapidly to AIDS. Importantly, no significant differences were found between depleted and undepleted RMs in terms of (i) availability of target cells; (ii) levels of SIV-specific CD8+ T cell responses; and (iii) SIV-specific humoral responses. Interestingly, peak viral load in depleted animals was associated with a striking increase in virus production by macrophages and dendritic cells.

Conclusion: These data indicate an essential role for CD4+ T-cells in mediating post-peak decline of viremia in SIV-infected RMs that may be associated with an ability to suppress virus production by macrophages. The complex balance between CD4+ T-cells as target cells and immune effectors may play a greater than anticipated role in the outcome of vaccine efficacy and should be considered during future vaccine design.

Background: The role of CD8+ T cells in containment of retroviral infections is well documented by studies of CD8+ T cell- mediated viral escape. It contrast, the immunological role of retrovirus-specific CD4+ T cells remains unclear, particularly in the setting of elite control. Here, we describe viral escape from CD4+ T cells concomitant with viral breakthrough and loss of elite control.

Methods: A SIVmac239-infected Indian rhesus macaque maintaining elite control of viremia (<1,000 vRNA copies/mL plasma) spontaneously lost control, experienced breakthrough viremia, and eventually succumbed to AIDS. We performed whole genome sequencing on plasma virus prior to the virus becoming undetectable and immediately following breakthrough and loss of elite control. We then investigated the T cell response directed against areas in the viral proteome exhibiting sequence divergence between the pre- and post-breakthrough time points.

Results: Comparison of the pre- and post-breakthrough viral sequences revealed surprisingly few amino acid substitutions post-breakthrough. These substitutions occurred within two previously defined and three previously undescribed CD8+ T cell epitopes, confirming the critical role of CD8+ T cells in control of viral replication. Interestingly, we identified two mutations within Gag, V63A and D205E, which occurred within CD4+ T cell epitopes. Both of these mutations abrogated CD4+ T cell recognition. While the V63A mutation also abrogated recognition of an overlapping CD8+ T cell epitope, the D205E mutation was solely targeted by a CD4+ T cell response. We confirmed that no CD8+ T cell responses were targeting this region. Finally, we demonstrated that viruses bearing the GagD205E mutation escaped CD4+ T cell effector function in vitro.

Conclusion: Virus-specific CD4+ T cells play an active role in the containment of retroviruses and are likely essential for the maintenance of low-level viremia. How these CD4+ T cells persist and exert their anti-viral function during retroviral infection remains unclear and warrants further investigation.

Conclusion: Lytic granule loading of CD8+ T cells and efficient delivery of active GrB to SIV-infected targets are associated with control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. These findings suggest ICE is a correlate of control of viral replication in chronic SIV infection. They also suggest the use of cytotoxic capacity as a predictor of immunologic control in the vaccine setting should be tested.

OA09.02

ADCC Titers to HIV-Infected Cells Are Detectable in the Majority of Vaccine Recipients in the RV144 Trial

Background: A modest reduction in the rate of HIV-1 infection was observed among vaccine recipients in the RV144 trial who received a prime-boost vaccine regimen with recombinant canarypox and soluble gp120. Whereas virus-specific CD8+ T cell responses were largely undetectable, antibodies capable of binding to gp120 were detectable in nearly all vaccinated subjects. However, these antibodies were unable to neutralize primary HIV-1 isolates, suggesting that antibodies may have contributed to protection by non-neutralizing effector mechanisms.

Methods: To determine if vaccinated subjects made antibodies capable of directing antibody-dependent cell-mediated cytotoxicity (ADCC), we used a novel assay to measure ADCC titers against HIV-infected cells in pilot samples from the RV144 trial. This assay is based on the killing of an HIV-infected CD4+ T cell line by a CD16+ NK cell line in the presence of serial dilutions of plasma, thus allowing ADCC titers to be measured against virus infected cells expressing the native, oligomeric conformation of the HIV-1 envelope glycoprotein. Using this approach, ADCC titers in blinded plasma samples from 80 vaccine recipients and 20 placebo controls were measured at baseline (week 0) and at two weeks after the last boost (week 26) against target cells infected with the HIV-1 subtype AE isolate 92TH023.

Background: Genome-wide transcriptional studies in non-human primates have suggested that the quality and magnitude of early immune responses to SIV infection are associated with downstream disease progression. The aim of this study is to identify, monitor, and interpret the whole blood transcriptional signature of acute HIV-1 infection (AHI).

Methods: A total of 56 AHI patients and matching healthy controls from both Africa and the United States were distributed into training, test, and validation datasets. Whole blood from these subjects was obtained for transcriptional profiling using microarrays. Both gene and modular transcriptional framework analyses were utilized to interpret the signature of AHI and for comparisons with other disease signatures including: influenza, tuberculosis, sepsis, and chronic HIV following interruption of anti-retroviral therapy (ART). Additional samples were collected at 1, 2, 4, 12, and 24 weeks post-enrollment for training and test set AHI subjects, to establish the longevity of the signature in the presence or absence therapy.

Results: We identified a robust AHI signature with increased activity in: interferon, cell cycle, cytotoxic, plasma cell, and B-cell modules amongst others. This activity was unique when compared to signatures obtained from patients with other infections. Only interferon and cell cycle activity was observed in both AHI subjects and chronically infected patients following interruption of therapy. Notably, 20% of AHI patients were observed to express a quiescent signature that clustered independent of the highly active signature described above. We found that these patients had significantly lower viral set-points (p = 0.01) when compared to patients with the active signature. Finally, longitudinal analysis demonstrated that the active AHI signature can persist independent of viral load in ART treated patients and up to 6 months in the absence of therapy.

Conclusion: Transcriptional monitoring of early HIV infection offers both quantitative and qualitative assessments patient responses that may be predictive of long-term disease progression.

P09.12

Correlation Between AIDS Pathology and the Biased Nucleotide Composition of Lentiviruses

Background: AIDS results from generalized immune activation after years of chronic HIV replication. However, most African primate species naturally infected with lentiviruses remain healthy. Addressing this issue is crucial for understanding AIDS pathogenesis. Because the genomes of lentiviruses present a particularly biased nucleotide composition compared to that of their primate hosts, we wondered whether host recognition of biased viral nucleic acids could induce hyper-responsiveness to HIV and AIDS pathogenesis.

Methods: We compared the nucleotide composition (A/C/G/T frequencies) of pathogenic and non-pathogenic primate lentiviruses with that of their hosts for every infection described in the literature. We measured the nucleotide divergence by computing the Chi-2 distance between the A/C/G/T frequencies in the complete viral sequence and the coding sequences of each host organism.

Results: We found that primate lentiviruses having the most divergent nucleotide composition compared to their hosts induce AIDS, whereas less divergent lentiviruses cause non-pathogenic infections. Similarly, the relative pathogenicity of HIV-1 subtypes correlates with their nucleotide divergence to the human genome.

To understand this observation at a molecular level, we investigated the ability of HIV-1 RNA fragments to stimulate in vitro the synthesis of type I interferon (IFN-I). We found that the nucleotide divergence of RNA fragments strongly correlates with type-I IFN activity. Based on these observations, we designed a large-scale, nucleotide-optimized SIV sequence derived from the pathogenic clone SIVmac239. We produced the corresponding artificial virus in lymphocyte cell line and analysed its capacity to induce type-I IFN in different cellular models. This synthetic virus presented the same replicative capacity than WT virus and a reduced ability to induce type-I IFN.

Conclusion: Overall, these data suggest for the first time a direct link between the nucleotide composition of lentiviruses and their pathogenicity. They also describe the synthesis a novel artificial SIV harbouring an attenuated pathogenic potential.

Background: Due to the current dual-epidemic of HIV+/AIDS and TB, functional understanding of HIV+/MTB co-infection immunology is critical for furthering vaccine and therapeutic strategies against both pathogens. IL-10 is an anti-inflammatory cytokine, produced by lymphocytes and monocytes, that has been shown to down-regulate expression of anti-viral and anti-bacterial cytokines such as IFN-gamma, IL2 and TNF-alpha. Elevated IL-10 levels have been reported during chronic human HIV infection and ex vivo IL-10 receptor blockade in the same patients has been shown to increase HIV specific T cell function.

Conclusion: This study concluded that there is a synergistic upregulation of IL-10 in HIV+/TB active patients as compared to HIV and MTB mono-infection. Additionally, this study concludes that novel ex vivo enhancement of MTB and HIV specific CD4 T cell function is possible with IL-10Ra blockade in HIV+/LTBI subjects.

P17.06

Cryptic Epitopes from Alternative Reading Frames Restricted by HLAs Associated with a Good Prognosis Are Frequently Recognized in HIV Infection

Background: Cryptic epitopes (CE) are products of translation of alternate reading frames (ARF, 2 sense and 3 antisense) that are commonly targeted during HIV/SIV infection. To understand the full extent of contributions of HIV specific CE in HIV-1 pathogenesis, we performed mapping of the CE derived from ARFs of nine HIV-1 encoded proteins.

Results: We predicted 520 (B*27), 955 (B*57) and 1027 (B*5801) potential CE in each of the five ARFs of the nine HIV-1 encoded proteins. The antisense frames encoded a majority of these CE. Predicted peptides with >50% probability of being an epitope were synthesized; B*27 (N = 30), B*57 (N = 39) and B*58 (N = 90). Overall, 26% and 2% of CHI patients and seronegative donors respectively had CE responses (p = 0.0006, Fischer’s exact). The overall responder frequency for HLA specific CE response was 22% (range 6%-43%) with the highest recognition frequency noted for HLA-B*5801/B*57 patients (p = 0.005, Fischer’s exact). In our comprehensive mapping analysis, 17% patients targeted CE from gag/pol ARFs and all responders were either HLA-B*27, B*57 or B*5801. Among these, the most frequently targeted were pol 2 (31%) followed by gag 2 and 5 (25%) frames.

Conclusion: These data underscore the importance of CE targeting especially those that are presented by the so-called ‘‘protective alleles’’ in HIV-1 infection and hence have implications for vaccine design.

Background: Although HIV-specific CD4+ T-cells are preferentially infected, there is growing evidence that these cells play a pivotal role in control of viremia. However, little is known about the recognition and HLA-class II restriction of HIV-specific CD4+ T-cells in the setting of chronic or spontaneously controlled HIV-infection. Yet, this knowledge will be crucial for the induction of protective immunity in a prophylactic vaccine.

Results: HIV-specific CD4+ T-cell responses were detected in all patient subgroup yet the breadth of these responses was significantly expanded in HIV-controllers (p = 0.029). Most HIV- specific CD4+ T-cells targeted epitopes within Gag, Nef and gp120. Strikingly, we observed significant differences in the immunodominance profile between patient subgroups that distinguished not only elite controllers from rapid progressors, but also from viremic controllers. While elite controllers dominantly targeted a tight cluster of conserved epitopes within p24, chronic progressors preferentially targeted epitopes within the C1/C3 domain of gp120. Moreover, the ratio of Env- and Gag-specific responses was a clear indicator of viral control. A multivariate bootstrap analysis identified four distinct Gag epitopes that were associated with spontaneous control (Probability = 0.60–0.85), while a single gp120 peptide in C3 was associated with high viremia (Probability = 0.82). Detailed characterization showed that promiscuous binding to multiple HLA-DR alleles occurs frequently, and revealed that HLA-DRB1*0701 is associated with slow disease progression (Probability = 0.88).

Conclusion: Our data demonstrate that significant differences exist in protein targeting by HIV-specific CD4+ T-cells between controllers and progressors. We also identified distinct epitopes associated with viral control that are conserved and bind promiscuously to multiple HLA-class II alleles, which will be important for vaccine design.

Methods: To develop an animal model to understand these results we determined if rhesus macaques chronically infected with a host range mutant adenovirus type-5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag-Pol- Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques during a series of repeated penile SIVmac251 exposures.

Results: We found that 2 of 9 rhesus macaques infected with Ad5hr prior to immunization became SIV infected after penile exposure to 103 TCID50 of SIVmac251. In contrast none of the 34 unimmunized animals, including 8 Ad5 seropositive animals immunized with the empty vector; Ad5 seronegative animals immunized with the Ad5 SIV vaccine (n = 9) or empty vector (n = 9) and 8 naive control animals became SIV infected after penile exposure to the same dose of SIVmac251. At the lowest SIV exposure dose (103 TCID50), the risk of infection was greater for animals with Ad5 titers at the time they were immunized with the MRKAd5 SIVmac239 subtype Gag-Pol-Nef vaccine compared to all the other animal groups (p = 0.004 based on log rank test). Penile exposure to more concentrated virus inoculums produced similar rates of infection in all animals groups. The Ad5 SIV vaccine induced CD8+ T cell responses in approximately 70% monkeys, which is similar to the proportion of humans that responded to HIV-1 vaccine.

Conclusion: The results of the NHP study described here recapitulate the results of the STEP trial and demonstrate that the results of NHP studies using challenge viruses and routes of exposure designed to mimic the variety and complexity of HIV-1 sexual transmission can reflect the outcomes of AIDS vaccine efficacy trials.

Background: The genetic diversity of HIV antigens represents a paramount challenge in the development of vaccines. Here, we establish rationale for circumventing this obstacle by eliciting cellular immune responses against stable human endogenous retrovirus K (HERV-K) antigens. Protein-level expression of HERV-K antigens has not been observed in healthy tissues, but does occur in various malignancies. We hypothesized that HIV would disrupt control of HERV-K expression within infected cells, targeting them for elimination by HERV-K-specific CD8+ T-cells.

Methods: HERV-K expression was assessed by qPCR, western blot, immunohistochemistry and mass-spectrometry. HERV-K- specific T-cell responses were identified by ELISPOT and a HERV-K-Env-specific CD8+ T-cell was cloned by IFN-g capture. Additional HERV-K-specific T-cell lines and clones were obtained by a dendritic-cell based expansion method. Recognition and elimination of HIV-infected cells were assessed by flow cytometry.

Results: HIV-infection resulted in HERV-K Gag and Env protein expression in primary CD4+ T-cells. HERV-K Gag and Env specific CD8+ T-cells specifically responded to, and eliminated, HIV-infected cells in an MHC-I-restricted manner. This recognition was dependent upon Vif, however expression of Vif alone was insufficient to recapitulate a response. A HERV-K-Env-specific CD8+ T-cell clone displayed comprehensive elimination of cells infected with a panel of 23 diverse HIV-1 and HIV-2 primary isolates as well as with SIVmac251.

Conclusion: The de-repression of HERV-K expression in HIV-infected cells, for which Vif is necessary but insufficient, constitutes a marker of infection that can be targeted by HERV-K-specific CD8+ T-cells. The unprecedented breadth of reactivity of HERV-K-specific T-cell responses against diverse lentiviruses, both implied by the proposed mode of action and observed in the current study, comprises an enticing advantage over HIV-1-specific T-cell responses which could be exploited in the development of HERV-K-targeted vaccines to treat or prevent HIV infection. These data also provides rationale for considering a role for HERV-K-specific T-cell responses in natural control of HIV.

P18.26 LB

Immunological Correlates of Protection Against Acquisition of HIV-1 Infection in the iPrEx Trial

Background: Pre-exposure prophylaxis for HIV-1 prevention in men who have sex with men (MSM) has been shown to reduce overall HIV-1 incidence by 44%. We hypothesized that exposure to HIV-1 before enrollment could have stimulated cell mediated immunity, which contributed to the observed protection.

Methods: We studied immune responses in highly exposed MSM enrolled in the iPrEx study who either seroconverted (SC; N = 93) or remained seronegative throughout follow up for up to 132 weeks (ESN; N = 485). Pre-infection time-points from SC participants were matched with time-points from ESN controls and analyzed for HIV-1 specific T-cell responses to p24, Nef, Vif, integrase (Int), reverse transcriptase (RT), and protease (Prot) using a standard IFNg ELIspot assay. Non-parametric tests were used to assess statistical significance of the comparisons between responses in SC and ESN participants.

On Tuesday at the AIDS Vaccine 2011 conference in Bangkok, Barton Haynes described the initial results of a mammoth effort to uncover immune correlates of protection in the RV144 vaccine trial (the presentation is available online via the conference website). As reported in the New England Journal of Medicine in 2009, the trial demonstrated a very slight but statistically significant degree of efficacy in reducing HIV infection risk; there were 51 infections in the vaccine group compared to 74 in the placebo group, indicating the vaccine reduced HIV acquisition by 31.2% (95% confidence interval: 1.1 to 52.1; p=0.04). Of potential importance, the reduction in risk was greatest in the first year, during which there were 12 infections among vaccine recipients compared to 30 in the placebo group. Over the subsequent 2.5 years of follow up, there were an additional 39 infections in the former group and 45 in the latter (essentially no difference).

To investigate whether any vaccine-induced immune responses were associated with acquiring or resisting infection in the trial, Haynes led a large collaborative effort that prioritized six different immunological tests and then studied them in a case-control format, comparing samples from vaccine recipients who became infected to those who did not. Samples were taken at week 26, two weeks after the final booster immunization. Ten infections occurred among vaccine recipients prior to week 26, so the total number that could be included in the case control study was 41. The total number of uninfected vaccine recipients included for comparison was 205. The six immunological tests studied were:

Binding of IgA antibodies in plasma to a panel of 14 HIV Env proteins.

Avidity of IgG antibodies to one of the gp120 proteins included in the AIDSVAX vaccine used in the trial (derived from a Thai CRF01_AE HIV isolate codenamed A244).

Antibody-dependent cellular cytotoxicity (ADCC) against cells infected with another a Thai CRF01_AE HIV isolate codenamed 92TH023 (the gp120 protein from this isolate is included in the ALVAC vector used in the trial).

Neutralizing antibody responses against a panel of six “tier 1” (relatively easy to neutralize) HIV isolates.

Binding IgG antibodies to the V1/V2 loops of HIV Env “scaffolded” onto a gp70 protein from murine leukemia virus (MLV). This assay was selected based on evidence that it identifies antibodies capable of binding to HIV Env in its natural conformation.

CD4 T-cell responses, measured by the production of cytokines and cell surface markers in response to stimulation with peptides from the 92TH023 isolate.

Helpfully, two scientists involved in the work—Morgane Rolland and Peter Gilbert—explain the statistical methodology used to evaluate whether any of these measures correlated with HIV acquisition in a new paper (see abstract and link below; the journal has made the paper open access). The authors note that multiple comparisons are involved in the analyses, which is known to increase the possibility of obtaining significant results simply by chance. The standard statistical tool for addressing this possibility is called a Bonferroni correction, but it was not used in this case because the goal was to generate hypotheses about possible immune correlates for additional studies, as opposed to confirming associations definitively. Instead of the Bonferroni method, the scientists used an approach in which each result is assigned a “q-value,” which represents the estimated chance of a false positive.

Haynes revealed in Bangkok that two of the six measures showed statistically significant associations with HIV acquisition: binding of IgA antibodies in plasma to HIV Env, which was linked to an increased relative risk of HIV infection of 1.54 (p=0.027); in other words, vaccine recipients displaying this response at week 26 of the trial were 54% more likely to subsequently become infected than those without it. Conversely, the presence of binding IgG antibodies to the V1/V2 loops of HIV Env scaffolded onto the MLV gp70 protein was associated with a relative risk of 0.57 (p=0.015), indicating that vaccine recipients who showed this response at week 26 were 43% less likely to acquire HIV than those who did not. For both results, the q value was 0.08, meaning that there is an approximately 8% chance they were false positives. The analyses have been repeated and confirmed by a second, independent group of statisticians. Haynes noted that the presence of IgA antibodies to Env did not enhance HIV infection risk compared to placebo recipients; rather, vaccinated individuals with high levels of these responses experienced the same risk of infection as the unvaccinated group. The opposite pattern was true for binding IgG antibodies to the V1/V2 loops: compared to placebo recipients, vaccinated individuals with the highest levels of these antibodies were around 50% less likely to acquire HIV infection.

In discussing the findings, Haynes took pains to stress that the results are exploratory in nature because there was no pre-specified plan for studying immune correlates in the original RV144 protocol. He also emphasized that further work is needed in order to understand whether there is a causal relationship between these immune responses and the trial outcome. The reason why IgA antibodies to Env would be associated with reduced vaccine efficacy is not yet clear, but there is some suggestive evidence that these antibodies interfere with antibody-dependent cellular cytotoxicity (ADCC), one of the immunological mechanisms that could have been responsible for the apparent protection observed in the trial. Similarly, it is not yet known whether the binding IgG antibodies to the V1/V2 loops were directly responsible for protecting against HIV infection (whether via ADCC or some other mechanism). However, these responses did wane significantly in vaccine recipients over the six months after the final week 24 immunizations, which appears to track with the protective effect of vaccination being almost entirely concentrated in the first year of the trial.

While much work remains, these new results represent potentially crucial clues for researchers working to improve on the marginal efficacy observed in RV144. Most optimistically, they hint that it might be possible to push the efficacy threshold of similar vaccine regimens over 50% by fine-tuning the types of immune responses that are induced. Although the induction of broadly neutralizing antibodies against HIV is still thought to be necessary to achieve a highly efficacious vaccine, the development of a candidate offering greater than 50% protection would be a huge step forward.

Since the RV144 vaccine combination showed efficacy in a Phase III trial, it provides an opportunity to generate hypotheses about the immune responses necessary for protection against HIV-1 infection, and these results could help devise vaccine candidates with higher efficacy. Here we describe how researchers can determine the correlates of immune protection for an HIV/AIDS vaccine, particularly in the context of the RV144 trial, and we discuss the terminology used to describe correlates and surrogates.